Light and electron microscopic analysis of two divisions of the suprachiasmatic nucleus in the young and aged rat.

The suprachiasmatic nucleus (SCN) is a principal controller of mammalian circadian rhythms. However, in spite of documented disturbance of biological rhythms in old animals, few significant age-related changes have been observed in this nucleus. This study examined age-related differences in SCN volume, neuronal number, density, and ultrastructural features in the entire rat SCN and in its two divisions, the denser ventromedial (compacta) and less dense dorsolateral (dissipata). Light and electron microscopic morphometric techniques were utilized in weanlings (21-28 days), young adults (3-6 mo), and aged (30-36 mo) animals. The total SCN volume, as well as volumes of the compacta region, were significantly greater in young adult and aged rats than in weanlings. Thus, as the rat ages the SCN increases in total size. However, the dissipata region appears to decrease in volume while the compacta increases. Even though the total number of SCN neurons was quite constant in the three age groups, the number of neurons in the dissipata region was decreased significantly in the young adult and aged groups as compared to the weanling. Neurons in the compacta region were usually spindled-shaped with two dendritic processes, while oval to spheroidal cells with 3-4 processes predominated in the dissipata. Nuclei of SCN cells were often invaginated. In weanlings, more SCN neuronal nuclei had invaginated nuclei in the dissipata region (66%) compared to the compacta (37%). In the two older age groups of rats, a higher percentage of invaginated neuronal nuclei were found in both regions. However, more were still found in the dissipata (90%) compared to the compacta (72%), even though the number of these cells in the compacta doubled. Thus, there was a large increase in the number of invaginated nuclei, as well as the number of invaginations, in the young adult rats compared to the weanling group, and this increase persisted in aged rats. SCN neurons usually had nuclei surrounded by a thin perimeter of cytoplasm containing sparse mitochondria and granular endoplasmic reticulum, multiple Golgi regions, and a moderate number of free ribosomes. In weanlings, mitochondria contained dense cristae and the granular endoplasmic reticulum was relatively prominent. Degenerative ultrastructural changes which included mitochondrial enlargement/vacuolation, Golgi vacuolation, lysosome, and lipofuscin development occurred in less than 10% of young adult SCN cells, and were more frequently found in the dissipata. In aged, rats 30% of the neurons showed degenerative changes in the dissipata compared with 18% in the compacta. Degenerative changes appeared highly correlated with the degree of membrane folding.(ABSTRACT TRUNCATED AT 400 WORDS)

Fate of septohippocampal neurons following fimbria-fornix transection: a time course analysis.

Neurons in the medial septum (MS) and vertical limb of the diagonal band (vDB) undergo degenerative changes following transection of their axons. These changes have been well studied by histological techniques such as Nissl stains and immunocytochemistry. A dramatic loss of stained neurons occurs following axotomy and this has been interpreted as indicative of neuronal death. However, since the staining intensity and the size of affected neurons may be reduced by axotomy, it is possible that the apparent neuronal death may actually be due to a decrease in somal size or the ability to detect neurons by routine histological methods. The present study describes the effects of axotomy on MS and vDB neurons which have been labeled by hippocampal injections of the retrograde tracer, Fluoro-Gold (FG), prior to transection of the fimbria-fornix and supracallosal stria. The number of FG-labeled neurons in the MS decreased by 21% at three weeks, 36% at six weeks, and 31% at ten weeks after fimbria-fornix transection. The reduction was statistically significant at 6 and 10 weeks. The number of FG-labeled neurons in the vDB showed no reduction at three weeks but was decreased by 31% and 37% at six and ten weeks, respectively. This was statistically significant only at 10 weeks. By comparison, the number of neurons immunoreactive for choline acetyltransferase (ChAT) was reduced by 75-80% at these time points. The size (area and diameter) of FG-labeled somata decreased in both the MS and vDB within three weeks following fimbria-fornix transection and remained relatively constant at the six- and ten-week time points.(ABSTRACT TRUNCATED AT 250 WORDS)

A study of NADPH-diaphorase positive septohippocampal neurons in rat.

Retrograde transport of fluorescent tracers and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemical techniques were combined in a study of septohippocampal projections in the rat. The dorsal (DH) and ventral (VH) hippocampus were simultaneously injected with different tracers (Fast Blue or Fluoro-Gold). Histochemical procedures revealed many NADPH-d positive cells located in the medial septum and the horizontal limb of the diagonal band. In the medial septum, NADPH-d positive neurons were mostly located lateral to the midline region and some of these were double-labeled by the tracer injected into the VH. Also, NADPH-d positive cells were found in the horizontal diagonal band and some of these were double-labeled following injections into the DH. No fluorescence/NADPH-d double-labeled neurons were observed in other structures known to project to the hippocampus.

Topographic organization and collateralization of the projections of the anterior and laterodorsal thalamic nuclei to cingulate areas 24 and 29 in the rat.

Fast blue and Rhodamine microspheres were used to analyze the topography and collateralization of the limbic nuclei of the thalamus to 4 defined areas of the cingulate gyrus (anterior 24, posterior 24, anterior 29, and posterior 29). The anterodorsal nucleus and the anteroventral nucleus project to posterior area 24 and all of area 29. The anteromedial nucleus projects generally to both areas 24 and 29, but principally to anterior area 24. The laterodorsal nucleus projects only to area 29. The anterior and posterior portions of the thalamic nuclei topographically project to the anterior and posterior regions of the cingulate gyrus, respectively. Neurons in the medial region of the anterior nuclei project to the anterior area of the cingulate gyrus while neurons located in the lateral region project to posterior areas. Collaterals (9-13%) from the anteromedial nucleus project diffusely to areas 24 and 29, while collaterals from other nuclei project primarily to areas adjacent to each other. Modulation of limbic system functions probably occurs via these projection fibers. Furthermore, the idea appears to be reinforced that at least a dual and coupled function exists for these direct thalamocingulate connections.

Innervation of the superficial pineal of the rat using retrograde tracing methods.

Fast blue (FB), rhodamine microspheres (RH), horseradish peroxidase (HRP), and wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) were used as retrograde tracers to study the innervation of the rat superficial pineal gland (SP). One of the tracers was injected into the gland of each animal. All four retrograde tracers injected into the gland always labeled neurons in the superior cervical ganglia (SCG). No retrograde labeling was ever seen in the suprachiasmatic nuclei, paraventricular hypothalamic nuclei, lateral hypothalamus, habenular nuclei, amygdalar nuclei, or superior salivatory nuclei. Retrograde labeling was seen in the anterior hypothalamic nuclei, anterior thalamic nuclei, lateral geniculate bodies, and midbrain tectal structures when a tracer spread from the injection site to the overlying cortex, tectum, or commissures. Control studies included injection of tracer into the subarachnoid space around the SP or into structures adjacent to the SP. Only the injection of FB or WGA-HRP into the subarachnoid space labeled neurons in the SCG. This labeling was probably due to the spread of tracer to the choroid plexus. These results agree with recent work confirming the existence of a direct projection of the SCG into the interstitium around pinealocytes. The evidence does not substantiate an innervation originating in the habenular nuclei; the superior salivatory nuclei; or any other diencephalic, midbrain, pontine, or medullary structure.

Strain differences in circadian drinking behaviors of ethanol and water in rats.

Voluntary drinking responses to an alternating three-bottle, two-choice paradigm to tap water or to 5% ethanol were measured in adult male Fisher (Fshr; N = 14) and spontaneously hypertensive (SP; N = 16) rats for 6-9 days. All animals were singly caged and housed separately in isolation chambers. The animals received light from 0600 to 1800 (CST) daily (LD 12:12) or remained in constant darkness (DD) at room temperature (23 degrees C). Food was freely available. Water and ethanol bottles were changed daily, and volumes of the respective fluids consumed by each rat were measured. A dim red light (approximately 0.5 lux) was used in handling animals in the dark. SP rats demonstrated significant (P less than 0.05) circadian drinking patterns of water and ethanol consumption under LD and DD lighting conditions. Fshr rats, however, exhibited a circadian pattern (P less than 0.02) only with regard to water consumption under an LD 12:12 lighting schedule; they did not exhibit circadian patterns of drinking water (P less than 0.054) in DD or ethanol in LD (P less than 0.24) or in DD (P less than 0.67) conditions. Volumes of ethanol consumption were also greater (P less than 0.05) in SP rats than in Fshr rats. It is concluded that differences exist in circadian drinking behaviors for both water and ethanol intake in two strains of rats. Perhaps variation in circadian patterns is an evolutionary mechanism that programs behavior over an appropriate time span for differences in physiological needs of nocturnal animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of techniques for retrograde labeling using the rat's facial nucleus.

This comparison of retrograde labeling substances was designed to define parameters which would produce the largest number of labeled neurons. The tracers are, horseradish peroxidase (HRP), wheat germ agglutinin (WGA), cholera toxin subunit B (CTB), wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP), cholera toxin subunit B-horseradish peroxidase conjugate (CTB-HRP), Fast blue (FB), Diamidino yellow (DY), and Rhodamine microspheres (RH). The whiskerpad of 29 adult rats was injected with 50 microliters of one of these tracers. The animals were anesthetized and perfused with various concentrations of fixatives to determine the labeling efficiency of the tracers (number of neurons labeled irrespective of their individual mode(s) of transport). All labeled neurons were counted and the averages of the 5 largest counts from every third section of the facial nucleus were compared based on changes in the procedures. The largest number of neurons labeled occurred when WGA was used as the tracer. WGA-HRP and CTB were better than HRP and twice as effective as CTB-HRP. Fast blue and HRP were equally effective. DY did not label as many neurons as CTB-HRP. Rhodamine microspheres did not label any neurons of this peripheral system. Glutaraldehyde reduced the number of neurons labeled.

Bilateral lesions of suprachiasmatic nuclei affect circadian rhythms in [3H]-thymidine incorporation into deoxyribonucleic acid in mouse intestinal tract, mitotic index of corneal epithelium, and serum corticosterone.

Investigations into the role of the suprachiasmatic nuclei (SCN) in the coordination of circadian rhythms have presented differing results. Several reports have shown that ablation of the suprachiasmatic nuclei (SCNA) alters the phase and amplitude of rhythms but does not abolish them. The present study investigates the effect of SCNA on the rhythms in cell proliferation in various regions of the intestinal tract as measured by the incorporation of [3H]-thymidine into deoxyribonucleic acid, in the mitotic activity of the corneal epithelium, and in serum corticosterone levels. The study involved mice with verified lesions of the SCN (six to 13 mice per time point) and control groups of both sham-operated and unoperated mice (seven of each per time point). The mice were killed in groups that represented seven time points over a single 24 hr span (3 hr intervals with the 0800 hr sampled both at start and end of the series). The tissues examined were the tongue, esophagus, gastric stomach, and colon for DNA synthesis, the corneal epithelium for mitotic index, and blood serum for corticosterone level. The most consistent result of SCNA was a phase advance in the rhythms in cell proliferation in the tongue, esophagus, gastric stomach, colon, and corneal epithelium. A reduction in rhythm amplitude occurred in the tongue, esophagus, and corneal epithelium; however, there was an amplitude increase for the stomach, colon, and serum corticosterone. The mesor (rhythm-adjusted mean) was increased by SCNA in all tissues except the corneal epithelium. These findings further support the role of the suprachiasmatic nuclear area in the control of rhythms in cell proliferation and corticosterone production, by acting as a "phase-resetter" and as a modulator of rhythm amplitude.

Effects of hippocampal lesions on ethanol intake in mice.

The effects of bilateral electrolytic hippocampal lesions on voluntary ethanol selection were examined in five groups (n = 10 each) of male mice. Mice sustained lesions in the dorsal hippocampus (DH), central hippocampus (CH), ventral hippocampus (VH), were sham-operated (SH) or left unoperated. CH mice exhibited a preference for ethanol compared with all other groups (p less than 0.01). In a second experiment, ethanol preference threshold and 24-hour drinking patterns were examined in CH and SH mice. These data revealed a higher preference threshold in CH mice, but not difference in drinking activity periods compared with controls. The central hippocampal area may be important in ethanol ingestive behavior in mice.

Amplitude-reduction and acrophase-advance of circadian mitotic rhythm in corneal epithelium of mice with bilaterally lesioned suprachiasmatic nuclei.

Lesions of the suprachiasmatic nuclei (SCN) have been reported to disrupt or abolish circadian rhythms of motor activity and body temperature as well as of several neuroendocrine functions. This study was undertaken to determine any effect the SCN may have on the prominent circadian rhythm characteristic of the mitotic index of the corneal epithelium. Singly caged adult mice of both sexes were fed ad libitum and standardized to 12 hours of light alternating with 12 hours of darkness. Animals were divided into experimental and control groups. Later they were killed in subgroups at the time intervals of 0800, 1200, 1000, 0000, 0400 hour. Mitotic counts were determined on both right and left corneas of all mice. The data were plotted separately for both groups and reflect colony variation. We conclude that major lesioning of the SCN or tis efferent projections disturbed but did not abolish the rhythm. In animals with unilateral lesions of the SCN, the mitotic index was consistently lower in the ipsilateral cornea. The mitotic index for animals with lesions which missed the SCN or its efferent projections did not differ from controls. The nuclei appear to function as pace-resetters and amplifiers of the rhythm studied.

Nomifensine chronopharmacology, schedule-shifts and circadian temperature rhythms in di-suprachiasmatically lesioned rats--modeling emotional chronopathology and chronotherapy.

Some emotional disorders are associated with alterations of biological rhythm characteristics ('echronism'). Chronotherapy aims empirically to 1. optimize the kind and timing of conventional psychopharmacologic treatment and, need be, to use such old or new molecules in the rational endeavor to 2. correct (disease-determining) rhythm alteration directly. With respect to the first aim, a reduction by timing of undesired pharmacodynamic effects, as well as an amplification of empirically desired ones, can be dramatically illustrated by circadian rhythms in tolerance to many drugs affecting the central nervous system of rodents. A more rational approach is aimed at correcting ecchronism. The new antidepressant drug, nomifensine, achieves this task in rats with bilateral suprachiasmatic lesions, exhibiting in the telemetered core temperature an echronism of varying degrees. In this model system for the chronobiotic treatment of ecchronism, the properly timed administration of nomifensine speeds the adjustment of bilaterally (suprachiasmatically) lesioned rats to a shift in the synchronizing light-dark schedule. With methodologic provisions, notably for treatment timed by pertinent marker rhythms, nomifensine deserves clinical tests in psychochronotherapy.

The cingulate bridge between allocortex, isocortex and thalamus.

The Fink-Heimer silver impregnation and the autoradiographic methods were used to study the fiber projections of the cingulate cortex in the squirrel monkey. It was found that this cortex provides inputs to the straitum, thalamus and several areas of isocortex. Evidence was found for a number of fiber projections (1) Fibers from the anterior limbic area were traced to the central part of the head of the caudate nucleus, putamen, septum, dorsomedial nucleus of the thalamus, anterior hypothalamus and lateral basal nucleus of the amygdala. (2) Projections from the cingulate area were traced to the lateral part of the head of the caudate nucleus, putamen, and to the centromedian, anterior, lateral dorsal, and lateral ventral thalamic nuclei and to medial nuclei of the base of the pons. (3) There were porjections from the retrosplenial area of the anterior, lateral dorsal, dorsomedial, and posterior thalamic nuclei and lateral nuclei of the pons. These results indicate that most of the cingulate gyrus is an intermediate structure between the thalamus and overlying cortex. The anterior limbic area forms a bridge between the thalamus and other areas of the cingulate gyrus and the frontal cortex. (4) the retrosplenial area and the posterior part of the cingulate area bridge the adjacent visual snesory association cortex and pelvic areas of the snesory motor cortex, respectively. These areas of the cingulate gyrus project directly to the striatum as well as to the thalamus, structurally providing limbic system input to subcortical motor structures.

Effects of amygdaloid lesions on reproductive function of grouped caged mice.

Amygdalotomized, sham-operated and intact wild-derived house mice of both sexes were group-caged (8-10) or singly caged for 3 weeks. The weights of ovaries (p is less than 0.05) and uteri (p is less than 0.01) were less in intact and sham-operated grouped females compared to singly caged intact and amygdalotomized females. The weight of the reproductive organs in amygdalotomized grouped female mice was not different from singly caged controls. Body weights among amygdalotomized grouped females were heavier (p is less than 0.05) than singly caged controls. Seminal vesicle weights of intact and sham-operated grouped males were less (p is less than 0.05) than that of singly caged controls and grouped amygdalotomized males. Plasma LH values from the various groups coincided with the morphological data. These results support the hypothesis that increased population density impairs reproductive function in mammals. Perhaps the effects of decreased pituitary-gonadal function associated with overcrowding are mediated via amygdalar nuclei.

Baseline sleep-wake patterns in the pointer dog.

The 24-hr electrographic patterns (EEG, EOG, EMG) of six normal pointer dogs were recorded in a laboratory setting. Two states of sleep (slow-wave and rapid eye movement) and wakefulness (alert and drowsy) were identified. The total recording period comprised 44% of alert wakefulness, 21% of the drowsy state, while slow-wave sleep occupied 23% and REM sleep 12% of the time. The mean length of a REM sleep episode averaged 6 min and the mean REM sleep cycle was 20 min. The mean polycyclic sleep-wake cycle was 83 min. Sleep episodes averaged 45 min and the mean waking episode was 38 min. There was an average of two REM sleep episodes per sleep-wake cycle. The dog has a propensity to sleep over a 16-hr interval from 1300 to 0500 but the most sleep occurred between 2100 and 0400 hr during darkness.